Epothilons C and D, preparation and compositions

ABSTRACT

The present invention relates to epothilon derivatives and to their use.

[0001] The present invention relates generally to epothilon derivativesand to their use in the preparation of medicaments. The presentinvention relates especially to the preparation of epothilon derivativesof general formulae 1 to 7 shown hereinafter and to their use in thepreparation of therapeutic compositions and compositions for plantprotection.

[0002] In formulae 1 to 7 given above:

[0003] R=H, C₁-₄alkyl;

[0004] R¹, R², R³, R⁴, R⁵=

[0005] H, C₁₋₆alkyl,

[0006] C₁₋₆acyl-benzoyl,

[0007] C₁₋₄trialkylsilyl,

[0008] benzyl,

[0009] phenyl,

[0010] benzyl or phenyl each substituted by C₁₋₆alkoxy,

[0011] C₆alkyl, hydroxy or by halogen;

[0012] It also being possible for two of the radicals R¹ to R⁵ to occurtogether to form a group —(CH₂)_(n)— wherein n is from 1 to 6, and thealkyl and acyl groups contained in the radicals are straight-chain orbranched radicals;

[0013] Y and Z are either identical or different and each representshydrogen, halogen, such as F, Cl, Br or I, pseudohalogen, such as —NCO,—NCS or —N₃, OH, O-(C₁₋₆)acyl, O-(C₁₋₆)alkyl, O-benzoyl, Y and Z mayalso be the O atom of an epoxy group, epothilon A and B not beingclaimed, or one of the C—C bonds forms a C═C double bond.

[0014] In formula 3, X generally represents —C(O)—, —C(S)—, —S(O)—,—CR¹R²—, wherein R¹ and R² are as defined above, or —SIR₂— wherein R isas defined above.

[0015] In formula 4, X represents oxygen, NOR³, N—NR⁴R⁵ or N—NHCONR⁴R⁵,wherein the radicals R³ to R⁵ are as defined above.

[0016] In formula 5, X represents hydrogen, C₁₋₁₆alkyl, C₁₋₁₆acyl,benzyl, benzoyl or cinnamoyl.

[0017] For epothilons A and B, see DE-A-41 38 042.

[0018] Compounds according to general formula 1 can be obtained startingfrom epothilon A and B and from their 3-O- and/or 7-O-protectedderivatives by opening the 12,13-epoxy group. If hydrohalic acids areused for that purpose in a preferably non-aqueous solvent, there beingobtained the halohydrins X=Hal; Y=OH and Y=OH, Y=Hal. Protonic acids,such as, for example, toluenesulphonic acid and trifluoroacetic acid,result, in the presence of water, in 12,13-diols which are then acylated(e.g. with carboxylic acid anhydrides and pyridine ortriethylamine/DMAP) or alkylated (alkylhalides and silver oxide)according to standard processes. For that purpose, the 3- and 7-hydroxygroups may be protected temporarily in the form of a formate (removalwith NH₃/MeOH) or of a p-methoxybenzyl ether (removal with DDQ).

[0019] Compounds according to general formula 2 are obtainable fromepothilon A and B and also from their 3-O- and/or 7-O-protectedderivatives by reduction, for example with NaBH₄ in methanol. If 3-OHand/or 7-OH are protected reversibly, then after acylation or alkylationand removal of the protecting groups there may be obtained5-O-monosubstituted or 3,5- or 5,7-O-disubstituted derivatives ofgeneral formula 2.

[0020] Reactions of epothilon A and B with bifunctional electrophilicreagents, such as (thio)phosgene, (thio)carbonyldiimidazole, thionylchloride or dialkylsilyl dichlorides or bistriflates yield compounds ofgeneral formula 3. Pyridine, trialkylamines, optionally together withDMAP or 2,6-lutidine in an aprotic solvent serve as auxiliary bases inthe process. The 3,7-acetals of general formula 3 are produced bytransacetalisation, for example of dimethylacetals in the presence of anacid catalyst.

[0021] Compounds according to general formula 4 are obtained fromepothilon A and B or from 3-O- and/or 7-O-protected derivatives thereofby ozonolysis and reductive treatment, for example with dimethylsulphide. The C-16-ketones may then be converted into oximes, hydrazonesor semicarbazones in accordance with standard processes known to theperson skilled in the art. They are, moreover, converted intoC-16-/C-17-olefins by Wittig, Wittig-Horner, Julla or Petersenolefination.

[0022] The 16-hydroxy derivatives according to general formula 5 areobtainable by reduction of the C-16-keto group, for example with analuminium hydride or borohydride. If 3-OH and 7-OH are provided withsuitable protecting groups, the 16-hydroxy derivatives may be eitheracylated or alkylated. The 3-OH and 7-OH groups are freed, for example,in the case of O-formyl by NH₃/MeOH and, in the case ofO-p-methoxybenzyl, by DDQ.

[0023] The compounds of general formula 6 are obtained from derivativesof epothilon A and B, in which the 7-OH group has been protected by acylor ether groups, by, for example, formylating, mesylating or tosylatingthe 3-OH group and then eliminating it by treatment with a base, forexample DBU. The 7-OH group can be freed as described above.

[0024] Compounds of general formula 7 are obtained from epothilon A andB or from 3OH- and 7-OH-protected derivatives thereof by basichydrolysis, for example with NaOH in MeOH or MeOH/water. Preferablycompounds of general formula 7 are obtained from epothilon A or B orfrom 3-OH- or 7-OH-protected derivatives thereof by enzymatichydrolysis, especially with esterases or lipases. The carboxy group canbe converted to an ester with a diazoalkane after protection of the19-OH group by alkylation.

[0025] Moreover, compounds of formula 7 may be converted into compoundsof formula 1 by lactonisation in accordance with the methods ofYamaguchi (trichlorobenzoyl chloride/DMAP), Corey(aldrithiol/triphenylphosphine) or Kellogg (omega-bromic acid/caesiumcarbonate). Relevant working methods may be found in Inanaga et al. inBull. Chem. Soc. Japan, 52 (1979) 1989; Corey & Nicolaou in J. Am. Chem.Soc., 96 (1974) 5614; and Krulzinga & Kellogg in J. Am. Chem. Soc., 103(1981) 5183.

[0026] To prepare the compounds according to the invention, it is alsopossible to start from epothilon C or D, where, for the derivatisation,reference may be made to the derivatisation methods described above. The12,13-double bond may be selectively hydrogenated, for examplecatalytically or with diimine; or epoxidised, for example withdimethyldioxirane or with a peracid; or converted into a dihalide,dipseudohalide or diazide.

[0027] The invention relates also to compositions for plant protectionin agriculture, forestry and/or horticulture, consisting of one or moreof the above-mentioned epothilon derivatives or consisting of one ormore of the above-mentioned epothilon derivatives together with one ormore common carrier(s) and/or diluent(s).

[0028] Finally, the invention relates to therapeutic compositionsconsisting of one or more of the above-mentioned compounds or of one ormore of the above-mentioned compounds together with one or more commoncarrier(s) and/or diluent(s). These compositions may especiallydemonstrate cytotoxic activities and/or cause immunosuppression and/orbe used to combat malignant tumours; they are particularily preferred ascytostatic agents.

[0029] The invention is illustrated and described hereinafter in greaterdetail by the description of a number of selected embodiments.

EXAMPLES Example 1 Compound 1a

[0030] 20 mg (0.041 mmol) of epothilon A are dissolved in 1 ml ofacetone, 50 μl (0.649 mmol) of trifluoroacetic acid are added and thereaction mixture is stirred overnight at 50° C. The reaction mixture isworked up by adding 1M phosphate buffer pH 7 and extracting the aqueousphase four times with ethyl acetate. The combined organic phases arewashed with saturated sodium chloride solution, dried over sodiumsulphate and freed of solvent. The raw product is purified bypreparative layer chromatography (eluant: dichloromethane/acetone,85:15). Yield 4 mg (19%) of isomer I 4 mg (19%) of isomer II Isomer IR_(i) (dichloromethane/acetone, 85:15) 0.46 IR (film) ny = 3440 (m, b,sh), 2946 (s, sh), 1734 (vs), 1686 (m), 1456 (m), 1458 (m), 1375 (w),1256 (s, sh), 1190 (w, b, sh), 1071 (m, sh), 884 (w), 735 (w) cm⁻¹. MS(20/70 eV) m/e (%) = 493 (43 [M-H₂O]⁺), 394 (47) 306 (32), 206 (30), 181(40), 166 (72), 139 (100), 113 (19), 71 (19), 57 (24), 43 (24).Microanalysis C₂₆H₃₉O₆NS calc.: 493.2498 for [M-H₂O]⁺ found: 493.2478Isomer II R₁ (dichloromethane/acetone, 85:15) 0.22 IR (film) ny = 3484(s, b, sh), 2942 (vs, sh), 1727 (vs), 1570 (w), 1456 (m), 1380 (m), 1265(s), 1190 (w), 109 (m), 975 (w), cm⁻¹. MS (20/70 eV) m/e (%) = 493 (21[M-H₂O]⁺), 394 (12) 306 (46), 206 (37), 181 (63), 166 (99), 139 (100),113 (21), 71 (23), 57 (33), 43 (28). Microanalysis C₂₆H₃₉O₆NS calc.:493.2498 for [M-H₂O]⁺ found: 493.2475

Example 2 Compound 1b

[0031] 55 mg (0.111 mmol) of epothilon A are dissolved in 0.5 ml oftetrahydrofuran, 0.5 ml of 1N hydrochloric acid is added, and thereaction mixture is stirred at room temperature for 30 minutes. 1NPhosphate buffer pH 7 is then added and the aqueous phase is extractedfour times with ethyl acetate. The combined organic phases are washedwith saturated sodium chloride solution, dried over sodium sulphate andfreed of solvent. The raw product is purified by preparative layerchromatography (eluant: dichloromethane/methanol, 90:10). Yield: 19 mg(32%). R_(i) (dichloromethane/acetone, 90:10) 0.46 IR (film) ny = 3441(s, br, sh), 2948 (s, sh), 1725 (vs, sh), 1462 (m), 1381 (w), 1265 (m),1154 (w), 972 (m, br, sh) cm⁻¹. UV (methanol) lambda_(max) (lg epslion)= 210 (4.29), 248 (4.11) nm. MS (20/70 eV) m/e (%) = 529 (13 [M⁺]), 494(10) 342 (38), 306 (23), 194 (32), 164 (100), 140 (31), 113 (15), 57(16). Microanalysis C₂₆H₄₀O₆CINS calc.: 529.2265 for [M⁺], found:529.2280

Example 3 Compound 1c

[0032] 25 mg (0.047 mmol) of 12-chloro-13-hydroxy-epothilon A (1b) aredissolved in 1 ml of dichloromethane, and 29 mg (0.235 mmol) ofdimethylaminopyridine, 151 μl (1.081 mmol) of triethylamine and 20 μl(0.517 mmol) of 98% formic acid are added. The reaction mixture iscooled with ice/salt. When −15° C. has been reached, 40 μl (0.423 mmol)of acetic anhydride are added to the reaction mixture, which is stirredfor 70 minutes at −15° C. Since thin-layer chromatography shows that thereaction is not complete, a further 6 mg (0.047 mmol) ofdimethylaminopyridine, 7 μl (0.047 mmol) of triethylamine, 2 μl of 98%formic acid (0.047 mmol) and 4 μl (0.047 mmol) of acetic anhydride areadded to the reaction mixture, which is stirred for 60 minutes. Thereaction mixture is worked up by heating to room temperature, adding 1Mphosphate buffer pH 7 and extracting the aqueous phase four times withethyl acetate. The combined organic phases are washed with saturatedsodium chloride solution, dried over sodium sulphate and freed ofsolvent. The raw product is purified by preparative layer chromatography(eluant: dichloromethane/acetone, 90:10).

[0033] Yield: 5 mg (18%). R₁ (dichloromethane/acetone, 90:10): 0.67 IR(film) ny = 3497 (w, b, sh), 2940 (s, b, sh), 1725 (vs), 1468 (m, b,sh), 1379 (m), 1265 (s), 1253 (s), 1175 (vs), 972 (m, b, sh), 737(s)cm⁻¹. MS (20/70 eV) m/e (%) = 613 (9 [M⁺]), 567 (43), 472 (63), 382(23), 352 (21), 164 (100), 151 (33), 96 (31), 69 (17), 44 (26).Microanalysis C₂₉H₄₀O₆NSCi calc.: 613.2112 for [M⁺] found: 613.2131

Example 4 Compound 1d

[0034] 10 mg (0.020 mmol) of epothilon B are dissolved in 0.5 ml oftetrahydrofuran, 0.5 ml of 1N hydrochloric acid is added and thereaction mixture is stirred at room temperature for 30 minutes. 1MPhosphate buffer pH 7 is then added and the aqueous phase is extractedfour times with ethyl acetate. The combined organic phases are washedwith saturated sodium chloride solution, dried over sodium sulphate andfreed of solvent. The raw product is purified by preparative layerchromatography (eluant: dichloromethane/acetone, 85:15).

[0035] Yield: 1 mg (9%). R_(i) (dichloromethane/acetone, 85:15) 0.38 MS(20/70 eV) m/e (%) = 543 (3 [M⁺]), 507 (14) 320 (19), 234 (9), 194 (17),182 (23), 164 (100), 140 (22), 113 (14), 71 (13). MicroanalysisC₂₇H₄₂O₆NSCl calc.: 543.2421 for [M⁺], found: 543.2405

Example 5 Compound 2a

[0036] 100 mg (0.203 mmol) of epothilon A are dissolved in 4 ml oftetrahydrofuran/1M phosphate buffer pH 7 (1:1), and sodium borohydride(150 mg=3.965 mmol) is added until the starting material has reactedcompletely according to thin-layer chromatography. Dilution with 1Mphosphate buffer pH 7 is then carried out and the aqueous phase isextracted four times with ethyl acetate. The combined organic phases arewashed with saturated sodium chloride solution, dried over sodiumsulphate and freed of solvent. The raw product is purified by silicachromatography (eluant: dichloromethane/acetone, 95:5—gradient—todichloromethane/acetone, 85:15). Yield (20%) R_(i)(dichloromethane/acetone, 75:25) 0.27 IR (film) ny = 3413 (s, b, sh),2965 (vs, sh), 1734 (vs), 1458 (m, b, sh), 1383 (m, sh), 1264 (s, b,sh), 1184 (m, b, sh), 1059 (s, sh), 966 (s), 885 (w), 737 (m) cm⁻¹. MS(20/70 eV) m/e (%) = 495 (6 [M⁺]), 477 (8) 452 (12), 394 (9), 364 (16),306 (49), 194 (19), 178 (35), 164 (100), 140 (40), 83 (21), 55 (27).Microanalysis C₂₆H₄₁O₈NS calc.: 495.2855 for [M⁺] found: 495.2623

Example 6 Compound 3a-d (a-d are Stereoisomers)

[0037] 100 mg (0.203 mmol) of epothilon are dissolved in 3 ml ofpyridine, 50 μl (0.688 mmol) of thionyl chloride are added and thereaction mixture is stirred at room temperature for 15 minutes. 1MPhosphate buffer pH 7 is then added and the aqueous phase is extractedfour times with ethyl acetate. The combined organic phases are washedwith saturated sodium chloride solution, dried over sodium sulphate andfreed of solvent. The raw product is purified and the four stereoisomers3a-d are separated by preparative layer chromatography (eluant:toluene/methanol, 90:10). Compound 3a Yield 4 mg (12%) R_(i)(toluene/methanol, 90:10) 0.50 IR (film) ny = 2961 (m, b, sh), 1742(vs), 1701 (vs), 1465 (m, sh), 1389 (m, sh), 1238 (s, sh), 1210 (vs,sh), 1011 (s, sh), 957 (s, b, sh), 808 (m, sh), 768 (s, sh), cm⁻¹. UV(methanol) lambda_(max) = 210 (4.50), 248 (4.35) nm. (lg epsilon) MS(20/70 eV) m/e (%) = 539 (40 [M⁺]), 457 (22) 382 (16), 318 (27), 222(30), 178 (30), 164 (100), 151 (43), 96 (38), 69 (29), 55 (28), 43 (20).Microanalysis C₂₆H₃₇O₇NS₂ calc.: 539.2011 for [M⁺] Compound 3b Yield 14mg (13%) R_(i) (toluene/methanol, 90:10) 0.44 IR (film) ny = 2963 (s,br, sh), 1740 (vs), 1703 (s), 1510 (w), 1464 (m, br, sh), 1389 (m, sh),1240 (s, br, sh), 1142 (m), 1076 (w), 1037 (w), 1003 (m), 945 (s, br,sh), 806 (m, sh), 775 (s), 737 (m) cm⁻¹. UV (methanol) lambda_(max) =211 (4.16), 250 (4.08) nm. (lg epsilon) MS (20/70 eV) m/e (%) = 539 (27[M⁺]), 475 (17) 322 (41), 306 (67), 222 (16), 206 (17), 194 (19), 178(32), 164 (100), 151 (33), 125 (18), 113 (15), 96 (39), 81 (23), 64(58), 57 (42), 41 (19). Microanalysis C₂₅H₃₇O₇NS₂ calc.: 539.2011 for[M⁺] found: 539.1998 Compound 3c Yield 4 mg (13%) R_(i)(toluene/methanol, 90:10) 0.38 MS (20/70 eV) m/e (%) = 539 (51 [M⁺]),322 (22) 306 (53), 222 (36), 178 (31), 164 (100), 151 (41), 96 (25), 81(20), 69 (26), 55 (25), 41 (25). Microanalysis C₂₆H₃₇O₇NS₂ calc.:539.2011 for [M⁺] found: 539.2001 Compound 3d Yield 1 mg (1%) R_(i)(toluene/methanol, 90:10) 0.33 MS (20/70 eV) m/e (%) = 539 (69 [M⁺]),322 (35) 306 (51), 222 (41), 178 (31), 164 (100), 151 (46), 96 (31), 81(26), 69 (34), 55 (33), 41 (35). Microanalysis C₂₆H₃₇O₇NS₂ calc.:539.2011 for [M⁺] found: 539.1997

Example 7 Compound 4a

[0038] 10 mg (0.020 mmol) of epothilon A are dissolved in 2 ml ofdichloromethane, cooled to −70° C. and then treated with ozone for 5minutes until there is a slight blue coloration. 0.5 ml of dimethylsulphide is subsequently added to the resulting reaction mixture, whichis heated to room temperature. The reaction mixture is worked up byfreeing it of solvent and finally by preparative layer chromatography(eluant: dichloromethane/acetone/methanol, 85:10:5). Yield 5 mg (64%)R_(i) (dichloromethane/acetone/methanol, 0.61 85:10:5) IR (film) ny =3468 (s, br, sh), 2947 (s, br,sh), 1734 (vs, sh), 1458 (w), 1380 (w),1267 (w), 1157 (w), 982 (w) cm⁻¹. UV (methanol) lambda_(max) = 202(3.53) nm. (lg epsilon) MS (20/70 eV) m/e (%) = 398 (2 [M⁺]), 380 (4)267 (14), 249 (17), 211 (20), 193 (26), 171 (34), 139 (34), 111 (40), 96(100), 71 (48), 43 (50). Microanalysis C₂₁H₃₄O₇ calc.: 398.2305 for [M⁺]found: 398.2295

Example 8 Compound 6a

[0039] 10 mg (0.018 mmol) of 3,7-di-O-formyl-epothilon A are dissolvedin 1 ml of dichloromethane, 27 μl (0.180 mmol) of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) are added and the reactionmixture is stirred at room temperature for 60 minutes. The reactionmixture is worked up by adding 1M sodium dihydrogen phosphate buffer pH4.5 and extracting the aqueous phase four times with ethyl acetate. Thecombined organic phases are washed with saturated sodium chloridesolution, dried over sodium sulphate and freed of solvent. After thesolvent has been removed, the resulting raw product is dissolved in 1 mlof methanol, 200 μl of an ammoniacal methanol solution (2 mmol NH₃/mlmethanol) are added and the mixture is stirred overnight at roomtemperature. For separation, the solvent is removed in vacuo. Yield 4 mg(22%) R_(i) (dichloromethane/acetone, (85:15) 0.46 IR (film) ny = 3445(w, br, sh), 2950 (vs, br, sh), 1717 (vs, sh), 1644 (w), 1466 (m, sh),1370 (m, sh), 1267 (s, br, sh), 1179 (s, sh), 984 (s, sh), 860 (w), 733(m) cm⁻¹. UV (methanol) lambda_(max) = 210 (4.16) nm. (lg epsilon) MS(20/70 eV) m/e (%) = 475 (28 [M⁺]), 380 (21) 322 (37), 318 (40), 304(66), 178 (31), 166 (100), 151 (29), 140 (19), 96 (38), 81 (20), 57(26). Microanalysis C₂₆H₃₇O₅NS calc.: 475.2392 for [M⁺] found: 475.2384

Example 9 Compound 6b

[0040] 50 mg (0.091 mmol) of 3,7-di-O-formyl-epothilon A are dissolvedin 1 ml of dichloroethane, 2 ml (0.013 mmol) of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) are added and the reactionmixture is stirred for 12 hours at 90° C. The reaction mixture is workedup by adding 1M sodium dihydrogen phosphate buffer pH 4.5 and extractingthe aqueous phase four times with ethyl acetate. The combined organicphases are washed with saturated sodium chloride solution, dried oversodium sulphate and freed of solvent. The raw product, consisting of twocompounds, is purified by preparative layer chromatography (eluant:dichloro-methane/acetone, 90:10). Yield 7 mg (15%) Substance code R_(i)(dichloromethane/acetone, (90:10) 0.62 IR (film) ny = 2951 (m, br, sh),1723 (vs), 1644 (w, br, sh), 1468 (w), 1377 (w), 1271 (m, br, sh), 1179(s), 987 (m, br, sh), 735 (w, br, sh) cm⁻¹. UV (methanol) lambda_(max) =210 (4.44) nm. (lg epsilon) MS (20/70 eV) m/e (%) = 503 (68 [M⁺]), 408(58) 390 (32), 334 (25), 316 (34), 220 (21), 206 (27), 194 (20), 181(33), 164 (100), 151 (34), 139 (28), 113 (20), 96 (82), 81 (33), 67(24), 55 (26), 43 (22) Microanalysis C₂₇H₃₇O₆NS calc.: 503.2342 for [M⁺]found: 503.2303

Example 10 Compound 6c

[0041] 5 mg (0.009 mmol) of 3,7-di-O-acetyl-epothilon are dissolved in 1ml of methanol, 150 μl of an ammoniacal methanol solution (2 mmol NH₃/mlmethanol) are added and the reaction mixture is stirred overnight at 50°C.

[0042] For separation, the solvent is removed in vacuo. The raw productis purified by preparative layer chromatography (eluant:toluene/methanol, 90:10). Yield 3 mg (67%) R_(i)(dichloromethane/acetone, (90:10) 0.55 IR (film) ny = 2934 (s, b, sh),1719 (vs, b, sh), 1641 (m), 1460 (m, sh), 1372 (s, sh), 1237 (vs, b,sh), 1179 (s, sh), 1020 (s), 963 (s, sh), 737 (vs) cm⁻¹. UV (methanol)lambda_(max) 210 (4.33) nm. (lg epsilon) = MS (20/70 eV) m/e (%) = 517(57 [M⁺]), 422 (58), 318 (31), 194 (20), 181 (34), 166 (100), 151 (31),96 (96), 81 (32), 69 (27), 55 (29), 43 (69). Microanalysis C₂₆H₃₈O₆NScalc.: 517.2498 for [M⁺] found: 517.2492

Example 11 Compound 7a

[0043] 20 mg (0.041 mmol) of epothilon are dissolved in 0.5 ml ofmethanol, 0.5 ml of 1N sodium hydroxide solution is added and thereaction mixture is stirred at room temperature for 5 minutes.

[0044] The reaction mixture is worked up by adding 1M phosphate bufferpH 7 and extracting the aqueous phase four times with ethyl acetate. Thecombined organic phases are washed with saturated sodium chloridesolution, dried over sodium sulphate and freed of solvent. The rawproduct is purified by preparative layer chromatography (eluant:dichloromethane/methanol, 85:15). Yield: 11 mg (52%) R_(F)(dichloromethane/methanol, 85:15): 0.92 IR (film): ny = 3438 (s, br,sh), 2971 (vs, br, sh), 1703 (vs), 1507 (m), 1460 (s, sh), 1383 (m, sh),1254 (w), 1190 (w, br, sh), 1011 (w, br, sh), 866 (w, br), 729 (s) cm⁻¹,MS (20/70 eV): m/e (%) = 423 (0.1 [M⁺]), 323 (4), 168 (89), 140 (100),85 (31), 57 (67), Microanalysis: C₂₃ H₃₇O₄NS calc.: 423.2443 for [M⁺]found: 428.2410

Example 12 Compound 7b

[0045] 5 mg (0.009 mmol) of 7-O-acetyl-epothilon are dissolved in 1 mlof methanol, 200 μl of an ammoniacal methanol solution (2 mmol NH₃/mlmethanol) are added and the reaction mixture is stirred at 50° C. fortwo days. For separation, the solvent is removed in vacuo. The rawproduct is purified by preparative layer chromatography (eluant:toluene/methanol, 90:10). Yield: 3 mg (59%) R_(I)(dichloromethane/methanol, 90:10): 0.63 IR (film): ny = 3441 (m, b, sh),2946 (s, sh), 1732 (vs), 1800 (w), 1451 (m), 1375 (m), 1246 (s, b, sh),1013 (m, b, sh) cm⁻¹, UV (methonal): lambda_(max) 211 (3.75). 247 (3.69)nm. (Ig epsilon) = MS (20/70 eV): m/e (%) = 567 (1 [M⁺]), 465 (4), 422(7), 388 (5), 194 (5), 182 (7), 168 (65), 164 (17), 140 (100), 97 (10),71 (22), 43 (27). Microanalysis: C₂₈ H₄₅O₆NS calc.: 587.2868 for [M⁺]found: 567.2849

Example 13

[0046] 50 mg of epothilon A are dissolved in 20 μl of dimethylsulphoxide and diluted with 30 ml of phosphate buffer (pH 7.1, 30 mM).After the addition of 5 mg of pig liver esterase (Boehringer Mannheim),the mixture is stirred for 2 days at 30° C. The mixture is acidified topH 5 with 2N HCl and the epothilonic acid 7 is extracted with ethylacetate. The organic phase is dried with sodium sulphate andconcentrated to dryness by evaporation in vacuo. Yield 48 mg (96%).

Example 14

[0047] 48 mg of epothilonic acid 7 are dissolved in 6 ml of abs. THFand, with stirring, 40 μl of triethylamine and 16 μl of2,4,6-trichlorobenzoyl chloride are added. After 15 minutes, theprecipitate is removed by filtration and the filtrate is added dropwise,within a period of 15 minutes, with rapid stirring, to a boilingsolution of 20 mg of 4-dimethylaminopyridine in 200 ml of abs. toluene.After a further 10 minutes, the mixture is concentrated by evaporationin vacuo and the residue is partitioned between ethylacetate/citrate-buffer (pH 4). After separation by preparative HPLC, theevaporation residue of the organic phase yields 15 mg of epothilon A.

Example 15 Epothilons C and D as Starting Materials

[0048] A. Production strain and culture conditions corresponding to theepothilon basic patent.

[0049] B. Production using DSM 6773

[0050] 76 liters of culture are grown as described in the basic patentand are used for inoculation in a production fermenter containing 700liters of production medium consisting of 0.8% starch, 0.2% glucose,0.2% soya flour, 0.2% yeast extract, 0.1% CaCl₂×2H₂O, 0.1% MgSO₄×7H₂O, 8mg/liter of Fe-EDTA, pH=7.4 and optionally 15 liters of Amberlite XAD-16adsorber resin. Fermentation takes 7 to 10 days at 30° C., with aerationwith 2 m³ air/h. The pO₂ is maintained at 30% by regulating the rotaryspeed.

[0051] C. Isolation

[0052] The adsorber resin is separated from the culture using a 0.7 m²100-mesh process filter and is freed of polar impurities by washing with3 bed volumes of water/methanol 2:1. Elution with 4 bed volumes, ofmethanol yields a raw extract which is concentrated by evaporation invacuo until the aqueous phase occurs. That is then extracted three timeswith the same volume of ethyl acetate. Concentration of the organicphase by evaporation yields 240 g of raw extract which is partitionedbetween methanol and heptane in order to separate lipophilic impurities.From the methanolic phase there are obtained by evaporation in vacuo 180g of isolate which is fractionated in three portions over Sephadex LH-20(20×100 cm column, 20 ml/min. methanol). The epothilons are contained inthe fraction which is eluted in the retention time from 240 to 300minutes and which comprises a total of 72 g. To separate the epothilons,chromatography is carried out in three portions on Lichrosorb RP-18 (15μm, 10×40 cm column, eluant 180 ml/min methanol/water 65:35). Afterepothilon A and B there are eluted epothilon C at R_(t)=90-95 ml andepothilon D at R_(t)=100-110 min, which are obtained, after evaporationin vacuo, in each case in a yield of 0.3 g of a colourless oil.

[0053] D. Physical properties

[0054] Epothilon C R=H

[0055] Epothilon D R=CH₃

[0056] Epothilon C

[0057] C₂₈H₃₉NO₅S[477]

[0058] ESI-MS: (positive ions): 478.5 for [M+H]⁺

[0059] 1H and 130, see NMR table

[0060] TLC: R_(t)=0.82

[0061] TLC aluminium foil 60 F 254 Merck, eluant:dichloromethane/methanol=9:1

[0062] Detection: UV extinction at 254 nm. Spraying withvanillin/sulphuric acid reagent, blue-grey coloration on heating to 120°C.

[0063] HPLC: R_(t)=11.5 min

[0064] Column: Nucleosil 100 C-18 7 μm, 126×4 mm.

[0065] Eluant: methanol/water=65:35

[0066] Flow rate: 1 ml/min

[0067] Detection: diode array

[0068] Epothilon D

[0069] C₂₇H₄₁NO₅S [491]

[0070] ESI-MS: (positive ions): 492.5 for [M+H]⁺

[0071] 1H and 13C, see NMR table

[0072] TLC: R_(t)=0.82

[0073] TLC aluminium foil 60 F 254 Merck, eluant:dichloromethane/methanol=9:1

[0074] Detection: UV extinction at 254 nm. Spraying withvanillin/sulphuric acid reagent, blue-grey coloration on heating to 120°C.

[0075] HPLC: R_(t)=15.3 min

[0076] Column: Nucleosil 100 C-18 7 μm, 125×4 mm

[0077] Eluant: methanol/water=65:35

[0078] Flow rate: 1 ml/min

[0079] Detection: diode array TABLE ¹H and ¹³C NMR data of epothilon Cand epothilon D in [D₆]DMSO at 300 MHz Epothilon C Epothilon D δ δ δ δ Hatom (ppm) C atom (ppm) (ppm) C atom (ppm)  1 170.3  1 170.1  2-Ha 2.38 2 38.4 2.35  2 39.0  2-Hb 2.50  3 71.2 2.38  3 70.8  3-H 3.97  4 53.14.10  4 53.2  3-OH 5.12  5 217.1 5.08  5 217.4  6-H 3.07  8 45.4 3.11  644.4  7-H 3.49  7 75.9 3.48  7 75.5  7-OH 4.46  8 35.4 4.46  8 36.3  8-H1.34  9 27.6 1.29  9 29.9  9-Ha 1.15 10 30.0 1.14 10 25.9  9-Hb 1.40 1127.6 1.38 11 31.8* 10-Ha 1.15* 12 124.6 1.14* 12 138.3 10-Hb 1.35* 13133.1 1.35* 13 120.3 11-Ha 1.90 14 31.1 1.75 14 31.6* 11-Hb 2.18 15 76.32.10 15 76.6 12-H 5.38** 16 137.3 16 137.2 13-H 5.44** 17 119.1 5.08 17119.2 14-Ha 2.35 18 152.1 2.30 18 152.1 14-Hb 2.70 19 117.7 2.65 19117.7 15-H 5.27 20 164.2 5.29 20 164.3 17-H 6.50 21 18.8 6.51 21 18.919-H 7.35 22 20.8 7.35 22 19.7 21-H₃ 2.65 23 22.6 2.65 23 22.5 22-H₃0.94 24 16.7 0.90 24 16.4 23-H₃ 1.21 25 18.4 1.19 25 18.4 24-H₃ 1.06 2714.2 1.07 26 22.9 25-H₃ 0.90 0.91 27 14.1 26-H₃ 1.63 27-H₃ 2.10 2.11

Example 16 Epothilon A and 12,13-Bisepi-Epothilon A from Epothilon C

[0080] 50 mg of epothilon C are dissolved in 1.5 ml of acetone, and 1.5ml of a 0.07M solution of dimethyldioxirane in acetone are added. After6 hours' standing at room temperature, concentration by evaporation invacuo is carried out and separation is effected by preparative HPLC onsilica gel (eluant: methyl tert-butyl ether/petroleum ether/methanol33:66:1).

[0081] Yield:

[0082] 25 mg of epothilon A,

[0083] R_(t)=3.5 min (analyt. HPLC, 7 μm, 4×250 mm column, eluant seeabove, flow rate 1.5 ml/min) and

[0084] 20 mg of 12,13-bisepi-epothilon A,

[0085] R_(t)=3.7 min, ESI-MS (pos. ions)

[0086] m/z=494 [M+H]⁺

[0087]¹H-NMR in [D₄] methanol, selected signals: delta=4.32 (3-H), 3.79(7-H), 3.06 (12-H), 3.16 (13-H), 5.54 (15-H), 6.69 (17-H), 1.20 (22-H),1.45 (23-H).

[0088] 12,13-bisepi-epothilon A R=H

1. Epothilon derivative of formula 1

wherein R=H, C₁₋₄alkyl; R¹, R²=H, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl each substituted by C₁₋₆alkoxy, C₆alkyl, hydroxy or by halogen; and the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals, and Y and Z are either identical or different and each represents hydrogen, halogen, pseudohalogen, OH, O-(C₁₋₆)acyl, O-(C₁₋₆)alkyl or O-benzoyl, or together form the O atom of an epoxy group or one of the C—C bonds of a C═C double bond, epothilon A and B being excluded.
 2. Epothilon derivative of formula 2

wherein R=H, C₁₋₄alkyl; R¹, R², R³=H, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl each substituted by C₁₋₆alkoxy, C₆alkyl, hydroxy or by halogen; the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals; and Y and Z are as defined according to claim 1 .
 3. Epothilon derivative of formula 3

wherein R=H, C₁₋₄alkyl; R¹, R²=H, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl each substituted by C₁₋₆alkoxy, C₆alkyl, hydroxy or by halogen; the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals, and X generally represents —C(O)—, —C(S)—, —S(O)—, —CR¹R²— or —SiR₂—, wherein R, R¹ and R² are as defined above and R¹ and R² may also together form an alkylene group having from 2 to 6 carbon atoms; and Y and Z are as defined according to claim 1 .
 4. Epothilon derivative of formula 4

wherein R=H, C₁₋₄alkyl; R¹, R², R³, R⁴, R⁵=H, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl each substituted by C₁₋₆alkoxy, C₆alkyl, hydroxy or by halogen; the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals, X represents oxygen, NOR³, N—NR⁴R⁵ or N—NHCONR⁴R⁵, wherein the radicals R³ to R⁵ are as defined above and R⁴ and R⁵ may also together form an alkylene group having from 2 to 6 carbon atoms; and Y and Z are as defined according to claim 1 .
 5. Epothilon derivative of formula 5

wherein R=H, C₁₋₄alkyl; R¹, R²=H, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl each substituted by C₁₋₆alkoxy, C₆alkyl, hydroxy or by halogen; the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals, and X represents hydrogen, C₁₋₁₆alkyl, C₁₋₁₆acyl, benzyl, benzoyl or cinnamoyl, and Y and Z are as defined according to claim 1 .
 6. Epothilon derivative of formula 6

wherein R=H, C₁₋₄alkyl and R¹=H, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl each substituted by C₁₋₆alkoxy, C₆alkyl, hydroxy or by halogen; the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals; and Y and Z are as defined according to claim 1 .
 7. Epothilon derivative of formula 7

wherein R=H, C₁₋₄alkyl and R¹, R², R³, R⁴=H, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl each substituted by C₁₋₆alkoxy, C₆alkyl, hydroxy or by halogen; the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals; and Y and Z are as defined according to claim 1 .
 8. Process for the preparation of an epothilon derivative of formula 7 according to claim 7 , characterised in that epothilon A, epothilon B, a 3-OH-protected derivative thereof or a 7-OH-protected derivative thereof is (a) enzymatically hydrolysed, especially with an esterase or lipase, or (b) hydrolysed in an alkaline medium, especially with sodium hydroxide in a methanol/water mixture, and the epothilon derivative of formula 7 is obtained and isolated.
 9. Process for the preparation of an epothilon derivative of formula 1 according to claim 1 , characterised in that an epothilon derivative of formula 7 according to claim 7 or in the form of the product of the process according to claim 8 is converted (a) according to the Yamaguchi method, or (b) according to the Corey method, or (c) according to the Kellogg method to form the epothilon derivative of formula 1 and that conversion product is isolated.
 10. Process for the preparation of epothilon A and/or 12,13-bisepi-epothilon A, characterised in that epothilon C is epoxidised, especially with dimethyldioxirane or with a peracid.
 11. Process for the preparation of epothilon B and/or 12,13-bisepi-epothilon B, characterised in that epothilon D is epoxidised, especially with dimethyldioxirane or with a peracid.
 12. Composition for plant protection in agriculture and forestry and/or in horticulture, consisting of one or more of the compounds according to any one of the preceding claims or of one or more of these compounds together with one or more common carrier(s) and/or diluent(s).
 13. Therapeutic composition, especially for use as a cytostatic agent, consisting of one or more of the compounds according to one or more of claims 1 to 7 or of one or more of the compounds according to one or more of claims 1 to 7 together with one or more common carrier(s) and/or diluent(s). 